A vector for introducing and expressing an foreign gene in mammalian cells is not only an essential tool for the study of basic life science, but it has also played an important role in applying the results to practical use in industry (for example, large-scale production of drugs) and clinical practice (for example, gene therapy). Progress in genetic engineering technology after the later half of the 1970's facilitated the isolation and amplification of particular gene DNA fragments (gene cloning) using Escherichia coli and yeast. Cloned DNA has been used conventionally for gene transfer to mammalian cells. In common practice, an artificial expression unit containing the coding region of a gene to be expressed (cDNA) linked with a promoter and a poly A addition site which are functional in mammalian cells has been prepared, or E. coli plasmid (a maximum of about 20 kb, cyclic), cosmid (a maximum of about 40 kb, cyclic), a bacteria artificial chromosome (BAC, maximum 200 kb, cyclic), and a yeast artificial chromosome (YAC, maximum 1 Mb, linear) which contain a genomic DNA fragment containing an original promoter and a poly A addition site as well as the coding region have been prepared in cyclic or linear form, and these have been transferred into cells by transfection or injection. When the introduced vector DNA has no origin of replication derived from a mammal, expression of the introduced gene will become transient because it is incapable of replication in the host cells and will be omitted during cell divisions. If the vector has an origin of replication, it produces a number of copies in the cells temporarily; however, they will be omitted gradually in the absence of selection pressure due to unequal partition to daughter cells during cell divisions. Therefore, expression is transient in this case as well. It is possible to select cell lines which express introduced genes in a constitutive manner by introducing a drug resistant gene simultaneously and applying drug selection pressure, though the introduced gene is incorporated into the chromosome of the host cell (integration). Integration affects both the introduced gene and the host chromosomes. Genes in the host chromosome may be destroyed (Pravtcheva et al., Genomics (USA), Vol. 30, p. 529-544, 1995). For the introduced gene, the number of copies may not be controlled, the copies may be inactivated (Garrick et al., Nature Genet. (USA), Vol. 18, p. 56-59, 1998) or affected by the control sequence on the host chromosome into which the gene has been integrated (Dobie et al., P.N.A.S. (USA), Vol. 93, p. 6659-6664, 1996; Alami et al., Hum. Mol. Genet. (UK), Vol. 9, p. 631-636, 2000). Thus, there is a need for the development of a method of introducing a given number of gene copies without destroying the host chromosome. A solution to such problems is to construct an artificial chromosome capable of autonomous replication/partition in host cells from animals including humans and to introduce genes into animal cells using this as a vector.
(1) Construction of a Human Artificial Chromosome (HAC)
Construction of human artificial chromosomes (hereinafter referred to as “HAC”) available in animal cells has been attempted in order to generate a vector to express foreign genes and, in biological terms, to identify the structure required for autonomous replication/partition in cells. There are three types of approaches to constructing HACs, i.e. (A) bottom up approach, (B) use of spontaneous chromosome fragments and (C) top-down approach (a natural chromosome is trimmed).
(A) Bottom Up Approach
The DNA sequence which is necessary for autonomous replication/partition has been identified in E. coli and yeast, and an artificial chromosome that provides for a given number of copies in host cells has been established (BAC or YAC). Similarly, an attempt has been made to use the bottom up approach to establish a HAC by introducing a cloned DNA fragment of a known sequence into animal cells and assembling the DNA fragment. A drug resistant gene derived from a YAC which contains an alphoid sequence of about 100 kb, which is a component of the human chromosome centromere, and a human telomere sequence were added and introduced into human fibrosarcoma cell line HT1080 (Ikenno et al., Nature Biotech. (USA), Vol. 16, p. 431-439, 1998). For the drug resistance cell clone, artificial chromosomes capable of autonomous replication/partition have been established; however, it is not that the introduced DNA sequence itself is maintained in the cell, but that reconstitution by amplification has occurred, and the sequence structure maintained by the cell is not clear.
In addition, the objective of the above research was to establish a HAC, and no research has been done to insert foreign genes.
(B) Use of Spontaneous Chromosome Fragments
A chromosome itself is an aggregate of genes, and possesses the elements required for autonomous replication/partition. Microcell mediated chromosome transfer has allowed for using a chromosome or fragments thereof as a tool for gene transfer in order to introduce a giant gene on the order of Mb, which exceeds the capacity of existing cloning vectors such as YAC. Fragments of human chromosomes 14, 2 and 22 including an antibody gene were transferred into mouse embryonic stem cells, and results showed that chimeras were produced, the antibody gene was expressed in the mice, the human chromosome fragments were retained stably in the chimeras and transmitted to the following generations through germ lines (Tomizuka et al., Nature Genet. (USA), Vol. 16, p. 133-143, 1997; Tomizuka et al., P.N.A.S. (USA), Vol. 97, p. 722-727, 2000). This example demonstrated the effectiveness of using the chromosome carrying the gene to be expressed as a vector. However, it is not realistic to modify chromosomes for every target gene. Desirably, a chromosome vector serving as a base structure is provided into which a target gene is easily inserted in order to take advantage of chromosome fragments as a vector and increase their versatility.
To that end, an attempt was made to use natural chromosome fragments to express foreign genes. The introduction and functional expression of the IL-2 gene (cDNA) or CFTR gene (human genome DNA) using an irradiated chromosome fragment (5.5 Mb) derived from human chromosome 1 as a vector has been reported (see for example, Guiducci et al., Hum. Mol. Genet. (UK), Vol. 8, p. 1417-1424, 1999; Auriche et al., EMBO Rep. (UK), Vol. 2, p. 102-107, 2002.) Hamster fibroblasts (CHO) were used as the host. In introducing a target gene into the fragmented minichromosome, alphoid DNA was used based on the hope that it would be inserted into the centromere domain of human chromosome 1; however, no particular insertion site or the copy number was identified. IL-2 dependent mouse lymphoblast cells became multiplicable in the IL-2-independent manner as a result of cell fusion with the CHO cell that retained IL-2 minichromosome, indicating functional complementarity. In addition, release of chlorine ion by stimulation with cAMP was observed in the CHO cell which retained the CFTR minichromosome, and the release of chlorine ion was suppressed by addition of a CFTR inhibitor. These showed systems for the insertion/expression of foreign genes using chromosome fragments as a vector, but the structure was not made clear and the insertion of foreign DNA was not controlled.
Chromosome fragments (2-3 Mb) derived from an irradiated hybrid cell were retained stably in hamster cells, which contained the centromere and a portion of the long arm of human chromosome 1, and the SDHC (succinate dehydrogenase complex, subunit C) gene. The G418 resistance gene was inserted by homologous recombination at the SDHC region. X-ray cell fusion was performed with mouse cells (L and 3T3), giving G418 resistance hybrid cells (Au et al., Cytogenet. Cell Genet. (Switzerland), Vol. 86, p. 194-203, 1999). This HAC has unknown structure because it uses natural chromosome fragments. Homologous recombination was used to introduce foreign genes into the HAC in a site-specific manner, though this approach had low insertion efficiency and was unsuitable for general purposes. Because the micronucleate cell fusion method was not used, host chromosomes were also present in addition to the target chromosome fragment. This only suggested the idea of expressing foreign genes using chromosomes as a vector.
In addition, by random insertion of a loxP site into a natural chromosome fragment (cyclic), a foreign gene (hygromycin resistance gene) was inserted using reconstitution of the drug resistance gene (hprt) as an indicator (Voet et al., Genome Res. (USA), Vol. 11, p. 124-136, 2001). This circular chromosome includes the centromere of human chromosome 20 and a portion of chromosome 1 (p22 region); however, its sequence has not been identified because it is a natural fragment. A foreign gene was introduced by site-specific recombination with a Cre/loxP system, though its constitution is unknown because the insertion of loxP into the chromosome is randomly occurred. Meanwhile, transfer into mouse ES cells, production of chimeras, and transmission to the progeny by microcell fusion have been shown. Although the method of inserting a target gene into an artificial chromosome is simple excepting that a natural chromosome fragment was used and loxP sites were randomly inserted, using an aberrant chromosome from a patient (mild mental retardation) is problematic in terms of safety and impractical.
(C) Top Down Approach
When a natural chromosome fragment is transferred into cells, many genes from the transferred chromosome fragment other than the target gene will be expressed at the same time. In an experiment of mouse ES cells, it is known that stability varies depending on the human chromosome used, and the contribution of cells retaining introduced chromosome fragments in chimeras decreases as the chromosome fragment increases in size. It is supposed that extra genetic expression disturbs propagation of host cells retaining chromosome fragments. Therefore it is thought that introduced chromosome fragments may be retained at higher rates by removing extra genes through modification of chromosomes.
A technology to shorten a chromosome by introducing a cloned telomere sequence by homologous recombination (telomere truncation) has been described as a method for deleting part of a chromosome (Itzhaki et al., Nature Genet. (USA), Vol. 2, p. 283-287, 1992). However, somatic cells of most animal species have extremely low homologous recombination frequency so that a lot of effort is required to obtain recombinants. Use of chicken cell line DT40 with high frequency homologous recombination as a host enabled efficient chromosome modification (Kuroiwa et al., Nucleic Acids Res. (UK), Vol. 26, p. 3447-8, 1998). The human X chromosome was transferred into the DT40 cell line by the microcell fusion method followed by telomere truncation (Mills et al., Hum. Mol. Genet. (UK), Vol. 8, p. 751-761, 1999). A linear minichromosome of 2.4 Mb was established by removing the short and long arms. The minichromosome was retained stably in hamster and human cells, though the copy number varied. Although stability of HACs was confirmed, no foreign gene was introduced so as to use them as a vector.
In addition, the human Y-chromosome in hamster cells was shortened by telomere truncation to establish a minichromosome of about 4 Mb which was retained stably in host cells (Heller et al., P.N.A.S. (USA), Vol. 93, p. 7125-7130, 1996). This minichromosome was transferred into mouse ES cells by the microcell fusion method, but was unstable. When chimeric mice were generated because the derivative minichromosome which integrated the mouse centromere sequence by chromosome reconstitution acquired stability in ES cells (Shen et al., Hum. Mol. Genet. (UK), Vol. 6, p. 1375-1382, 1997), germ line transmission was confirmed (Shen et al., Curr. Biol. (UK), Vol. 10, p. 31-34, 2000). A chimeric chromosome was shown to be retained in mice, but its structure is unknown because of chromosome reconstitution and no research was done for the introduction/expression of foreign genes.
(2) Insertion of Foreign Genes into HACs
Similarly important to the establishment of HACs as vectors as described above is the establishment of a method for introducing a target gene into the HAC. However, as described above, the establishment of HACs itself has not yet been completed, and for the introduction of foreign genes, only random insertion of drug resistant genes has been suggested; besides no detailed analysis has been done.
Stability in mice and germ line transmission have been confirmed for the spontaneous fragment SC20 from human chromosome 14, which was isolated to generate a mouse retaining the human antibody heavy chain gene. A method (chromosome cloning) of cloning chromosome regions (regions of human chromosomes 2 and 22 including the antibody light chain genes) of the Mb order by reciprocal translocation was established which used the Cre/loxP system (Kuroiwa et al., Nature Biotech. (USA), Vol. 18, p. 1086-1090, 2000). This method was aimed at establishing the HAC of defined structure that contained no unnecessary genes, and it is effective when applied to giant genes of a size exceeding the capacity of other cloning vectors (for example, YAC), such as antibody genes.
In either case, no HAC vector system has been established to date which satisfies the conditions: 1) the structure has been identified and unnecessary genes have been removed, 2) the HAC vector can be maintained stably in cultured cells and individuals, and 3) foreign DNA can be easily introduced into it.